Spatial distribution and temporal change in cytosolic pH and [Ca 2+] in resting and activated single human platelets

Abstract

In human platelets, a rapid rise in cytoplasmic Ca 2+ and slower rise in cytoplasmic pH follow stimulation by thrombin. With the Ca 2+ probe Fura-2 and the pH probe SNARF-1 for digitized fluorescence microscopy, we studied simultaneously the distribution and changes with time of [pH] i and [Ca 2+] i in individual human platelets. In platelets coloaded with both probes, the probes had no detectable fluorescence at each other's excitation wavelength. The monovalent cation ionophore, nigericin (2 μM), produced a homogeneous rise in pH but no change in [Ca 2+]. Platelets, in contact with glass, spread and developed an irregular, apparently mutually independent rise in both [Ca 2+]and pH. Stimulation of platelets by thrombin 1.0 U/ml elevated [Ca 2+] i and produced slow alkalinization without initial acidification. Replacement of extracellular Na+ by choline abolished thrombin-induced alkalinization, but had no effect on thrombin-induced [Ca 2+]; elevation. ADP 10 μM caused a rapid rise of [Ca 2+] i and transient alkalinization. Most stimulated platelets developed a gradient in pH, that was highest in the center. ADP and thrombin caused oscillation of [Ca 2+]; but not of [pH] i. We conclude that alkalinization in stimulated platelets, presumably involving Na +/H + antiport, is not essential for the rise of [Ca 2+] i that may accompany it.